Antibodies directed against idiotypic determinants on anti-Staphylococcal nuclease antibodies from idfferent mouse strains have been produced in rats and in pigs. The idiotypes are detected by ELISA assays and by the inhibition of antibody-mediated inactivation of nuclease. By screening a variety of strains and offspring from appropriate matings between strains for the presence of idiotypes and other markers, it has been shown that idiotype expression is linked to the heavy chain allotype markers. By means of an in vitro-TNP nuclease plaque-forming cell response, idiotypic markers have been demonstrated on T helper cells. Administration of anti-idiotypic antibodies to mice has been found to induce idiotype expression in the serum of these animals. This effect appears to involve T cells, since it is not observed in nude mice, and since idiotype-bearing T helper cells for in vitro anti-TNP nuclease response have been found in spleens from such treated animals. Several hybridomas reactive with nuclease and/or anti-idiotype have been produced. Syngeneneic anti-idiotypes have also been produced and are presently being characterized in both antibody and T cell systems. Competitive binding studies are used to determine epitopes of nuclease as defined by available monoclonal antibodies. Site-directed mutagenesis of the nuclease gene has provided numerous point mutants of nuclease which are being studied for change in immune reactivity.